Method of reducing intraocular pressure in humans

ABSTRACT

Provided herein is a method of reducing intraocular pressure (IOP) in humans using N6-cyclopentyladenosine (CPA), CPA derivatives or prodrugs or enhanced cornea permeability formulations of CPA. In one embodiment, the invention is directed to CPA derivatives or prodrugs that are permeable to the cornea. In another embodiment, the invention is directed to uses of certain compounds in human subjects for reducing and/or controlling elevated or abnormally fluctuating IOPs in the treatment of glaucoma or ocular hypertension (OHT).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/552,160 filed Nov. 24, 2014, which is a continuation of U.S. patentapplication Ser. No. 13/909,288 filed Jun. 4, 2013, which issued as U.S.Pat. No. 8,895,530 on Nov. 5, 2014. U.S. patent application Ser. No.13/909,288 is a continuation of U.S. patent application Ser. No.13/072,349, filed Mar. 25, 2011, which issued as U.S. Pat. No. 8,476,247on Jul. 2, 2013, and which claims priority to U.S. ProvisionalApplication No. 61/318,105, filed Mar. 26, 2010. The contents of anypatents, patent applications, and references cited throughout thisspecification are hereby incorporated by reference in their entireties.

TECHNICAL FIELD OF THE INVENTION

Provided herein is a method of reducing intraocular pressure (IOP) inhumans using N6-cyclopentyladenosine (CPA), CPA derivatives or prodrugs,or enhanced cornea permeability formulations of CPA. In one embodiment,the invention is directed to CPA derivatives or prodrugs that arecornea-permeable. In another embodiment, the invention is directed touses of CPA compounds in human subjects for reducing and/or controllingelevated or abnormally fluctuating IOPs in the treatment of glaucoma orocular hypertension (OHT).

Cyclopentyladenosine-N⁶-cyclopentyladenosine

BACKGROUND OF THE INVENTION

Glaucoma refers to a group of optic neuropathies that are characterizedby loss of retinal ganglion cells and atrophy of the optic nerve withresultant visual field loss. The disease is the leading cause ofirreversible blindness worldwide and the second leading cause ofblindness, behind cataracts. Clinical trials have demonstrated thatelevated IOP is a major risk factor for glaucoma and have validated therole of lowering IOP in the management of glaucoma.

Glaucoma is classified according to three parameters: 1) the underlyingcause, i.e., primary (idiopathic) or secondary (associated with someother ocular or systemic conditions); 2) the state of the anteriorchamber angle, i.e., open angle (open access of the outflowing aqueoushumor to trabecular meshwork) or closed angle (narrow angle; thetrabecular meshwork is blocked by apposition of the peripheral iris andthe cornea); and 3) chronicity, i.e., acute or chronic. Althoughsecondary forms of glaucoma with clear etiologies do exist (e.g.,pseudoexfoliation and pigmentary dispersion), the most common form ofglaucoma is primary open angle glaucoma (POAG).

OHT is a condition in which IOP is elevated but no glaucomatous findingshave been observed (Bell, 2005). The Ocular Hypertension Studydemonstrated that patients with OHT have an overall risk of 10% over 5years of developing glaucoma and that this risk can be cut in half bythe institution of medical treatment that reduces IOP.

Drug therapies that have proven to be effective for the reduction ofintraocular pressure include both agents that decrease aqueous humorproduction and agents that increase the outflow facility. Such therapiesare in general administered by one of two possible routes: topically(direct application to the eye) or orally. However, pharmaceuticalocular anti-hypertension approaches have exhibited various undesirableside effects. For example, miotics such as pilocarpine can causeblurring of vision, headaches, and other negative visual side effects.Systemically administered carbonic anhydrase inhibitors can also causenausea, dyspepsia, fatigue, and metabolic acidosis. Certainprostaglandins cause hyperemia, ocular itching, and darkening ofeyelashes, irises, and periorbital tissues. Further, certainbeta-blockers have increasingly become associated with serious pulmonaryside-effects attributable to their effects on beta-2 receptors inpulmonary tissue. Sympathomimetics cause tachycardia, arrhythmia andhypertension. Such negative side-effects may lead to decreased patientcompliance or to termination of therapy such that normal visioncontinues to deteriorate. Additionally, there are individuals who simplydo not respond well when treated with certain existing glaucomatherapies.

Therefore, there remains a need for new treatments and therapies forelevated intraocular pressure (IOP), and conditions caused by elevatedIOP. There is also a need for compounds useful in the treatment orprevention or amelioration of one or more symptoms of elevated IOP andconditions caused by elevated IOP.

SUMMARY OF THE INVENTION

There remains a need for new treatments and therapies for elevatedintraocular pressure (IOP), and conditions caused by elevated IOP. Thereis also a need for compounds useful in the treatment or prevention oramelioration of one or more symptoms of elevated IOP and conditionscaused by elevated IOP.

In a first aspect, the present invention provides a method of reducingintraocular pressure comprising the step of: delivering an effectiveamount of cyclopentyladenosine according to Formula I to the anteriorchamber of an affected eye of a human,

with the proviso that the compound of Formula I is not delivered in theform of compound A

((2R,3S,4R,5R)-5-(6-(cyclopentylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methylnitrate

In another aspect the method as defined above further comprises theprior, simultaneous or sequential, application of a second IOP reducingagent. In one embodiment the second IOP reducing agent is selected fromthe group comprising: β-blockers, prostaglandin analog, prostamides,carbonic anhydrase inhibitors, rho-kinase inhibitors, α₂ agonists,miotics, neuroprotectants, A₁ agonist, A₃ antagonists, A₂A agonists andcombinations thereof.

In yet another aspect, the present invention provides a method ofreducing intraocular pressure comprising the step of: delivering aneffective amount of cyclopentyladenosine according to Formula I in acornea-permeable form to the anterior chamber of an affected eye of ahuman,

or a pharmaceutically acceptable salt thereof,

with the proviso that the compound of Formula I is not delivered in theform of compound A

((2R,3S,4R,5R)-5-(6-(cyclopentylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methylnitrate

In another aspect the method as defined above further comprises theprior, simultaneous or sequential, application of a second IOP reducingagent. In one embodiment the second IOP reducing agent is selected fromthe group comprising: β-blockers, prostaglandin analog, prostamides,carbonic anhydrase inhibitors, rho-kinase inhibitors, α₂ agonists,miotics, neuroprotectants, A₁ agonists, A₃ antagonists, A₂A agonists andcombinations thereof.

In another aspect the present invention is directed to a compound ofFormula II,

or a pharmaceutically acceptable salt thereof,

wherein R₁ is selected from —(CO)C₁-C₆ alkyl, —(CO)CH(halo)₂,—(CO)phenyl, or a —(CO)C₁-C₁₀ optionally branched aliphatic, —(CO)C₃-C₈cycloalkyl which is optionally substituted with one or more of hydroxyor —(CH₂)_(n)OH where n is 1-6, —(CO)aryl which is optionallysubstituted with one or more of hydroxy or —(CH₂)_(n)OH where n is 1-6;or a —(CO) C₃-C₇ heterocyclic which is optionally substituted with oneor more of hydroxy or —(CH₂)_(n)OH, where n is 1-6; R₂ is selected from—H or halo; and R₃ is selected from —H, hydroxy, —O(CO)CH(halo)₂,—O(CO)(CH₂)₂CH₃, —O(CO)CH(CH₃)₂, —O(CO)CH₂C(CH₃)₃.

In still another aspect the invention is directed to a method ofreducing IOP and associated diseases and conditions caused by elevatedIOP in a human subject by administering an effective amount of acompound of Formula II to an affected eye of the human subject,

or a pharmaceutically acceptable salt thereof,

wherein R₁ is selected from —(CO)C₁-C₆ alkyl, —(CO)CH(halo)₂,—(CO)phenyl, or a —(CO)C₁-C₁₀ optionally branched aliphatic, —(CO)C₃-C₈cycloalkyl which is optionally substituted with one or more of hydroxyor —(CH₂)_(n)—OH where n is 1-6, —(CO)aryl which is optionallysubstituted with one or more of hydroxy or —(CH₂)_(n)—OH where n is 1-6;or a —(CO) C₃-C₇ heterocyclic which is optionally substituted with oneor more of hydroxy or —(CH₂)_(n)—OH where n is 1-6; R₂ is selected from—H or halo; and R₃ is selected from —H, hydroxy, —O(CO)CH(halo)₂,—O(CO)(CH₂)₂CH₃, —O(CO)CH(CH₃)₂, —O(CO)CH₂C(CH₃)₃.

In one embodiment the diseases and conditions caused by elevated IOP ina human are selected from the group consisting of normal-tensionglaucoma, OHT, and POAG.

In one embodiment the method comprises the step of applying about 0.05mg/ml to about 7.0 mg/ml of a compound according to Formula II from 1 to4 times daily, or in another embodiment the method comprises the step ofapplying about 20-700 μg of a compound according to Formula II from 1 to2 times daily or in another embodiment the method comprises the step ofapplying about 350 μg of a compound according to Formula II from 1 to 2times daily.

In one embodiment the IOP of the affected eye is reduced by at least10%. In another embodiment the IOP of the affected eye is reduced by atleast 10-20%. In a further embodiment the IOP of the affected eye isreduced by 20% or more.

In one embodiment the IOP of the affected eye is reduced by at least 10%for more than 3 hours, in another embodiment the IOP of the affected eyeis reduced by at least 10-20% for more than 3 hours, in a furtherembodiment the IOP of the affected eye is reduced by 20% or more formore than 3 hours and in another embodiment the IOP of the affected eyeis reduced by at least 10% for at least 6 hours.

In another aspect the method as defined above further comprises theprior, simultaneous or sequential, application of a second IOP reducingagent. In one embodiment the second IOP reducing agent is selected fromthe group comprising: β-blockers, prostaglandin analog, prostamides,carbonic anhydrase inhibitors, rho-kinase inhibitors, α₂ agonists,miotics, neuroprotectants, ion channel modulators, A₁ agonists, A₃antagonists, A₂A agonists and combinations thereof.

In one embodiment the effective amount of the compound of Formula II isat least 20 μg.

In one embodiment the effective amount of the compound of Formula II isbetween 60 μg and 700 μg.

In one embodiment the effective amount of the compound of Formula II isadministered as a single dose.

In one embodiment the effective amount of the compound of Formula II isadministered as a twice daily dose.

In another aspect there is provided an ophthalmic pharmaceuticalcomposition comprising a compound of Formula II as defined above and apharmaceutically acceptable vehicle or excipient.

In one embodiment the pharmaceutically acceptable vehicle or excipientis selected from the group comprising of: ophthalmologically acceptablepreservatives, surfactants, viscosity enhancers, penetration enhancers,gelling agents, hydrophobic bases, vehicles, buffers, sodium chloride,and water.

In one embodiment the composition further comprises a second IOPreducing agent in addition to a compound of Formula I as defined above.The second IOP reducing agent is selected from the group comprising:β-blockers, prostaglandin analogs, prostamides, carbonic anhydraseinhibitors, rho-kinase inhibitors, α₂ agonists, miotics,neuroprotectants, ion channel modulators, A₁ agonists, A₃ antagonists,A₂A agonists and combinations thereof.

In another aspect there is provided a compound of Formula III

wherein R₄ is selected from —CH₃, —CH(CH₃)₂, —CH(halo)₂, —CH₂C(CH₃)₃,—CH₂CH(CH₃)₂, —C(CH₃)₃, —(CH₂)₂CH₃, —(CH₂)₃CH₃, —(CH₂)₄CH₃, —(CH₂)₅CH₃,—(CH₂)₆CH₃, —CH₂CH₃, -phenyl, or -benzyl.

In a further aspect CPA or a prodrug of CPA such as that of Formula IIor Formula III can be used to lower and/or control IOP associated withnormal-tension glaucoma, OHT, and POAG in humans. In certainembodiments, when used to treat normal-tension glaucoma or OHT, thecompounds of Formula II can be formulated in pharmaceutically acceptablecompositions suitable for topical delivery to the eye. Anotherembodiment of the present invention comprises an ophthalmicpharmaceutical composition useful in the reduction of intraocularpressure, comprising an effective amount of a compound according toFormula II.

It is to be further appreciated that the use of a compound of CPA or ofFormula II as defined above, or ophthalmic compositions as defined abovemay be used for manufacture of a medicament for reducing IOP in anaffected eye of a human subject. It is recognized that compounds ofFormula I, II or Formula III can contain one or more chiral centers.This invention contemplates all enantiomers, diastereomers, and mixturesof Formulas I, II and III thereof.

Furthermore, certain embodiments of the present invention comprisepharmaceutically acceptable salts of compounds according to Formula I,II or III.

Pharmaceutically acceptable salts comprise, but are not limited to,soluble or dispersible forms of compounds according to Formula I, II orIII that are suitable for treatment of disease without undue undesirableeffects such as allergic reactions or toxicity. Representativepharmaceutically acceptable salts include, but are not limited to, acidaddition salts such as acetate, citrate, benzoate, lactate, or phosphateand basic addition salts such as lithium, sodium, potassium, oraluminum.

The foregoing brief summary broadly describes the features and technicaladvantages of certain embodiments of the present invention. Furthertechnical advantages will be described in the detailed description ofthe invention that follows. Novel features which are believed to becharacteristic of the invention will be better understood from thedetailed description of the invention when considered in connection withany accompanying figures and examples. However, the figures and examplesprovided herein are intended to help illustrate the invention or assistwith developing an understanding of the invention, and are not intendedto be definitions of the invention's scope.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows a cross section diagram of a human eyeball and shows therelationship of the cornea to the anterior chamber.

FIG. 2: shows the CPA levels and Compound A levels detected in theplasma of a human subject after the time of administration of 350 μg ofCompound A topically to the cornea of the human subject relative to theIOP measured in the subject.

FIG. 3: shows diagrammatically the apparatus employed to determine thein vitro cornea permeability of CPA esters.

FIG. 4: shows the results from an in vivo study using Dutch Beltedrabbits whereby Compound A and CPA alone were administered topically tothe cornea of a subject eye of the rabbits and showing the subsequentbuild up in concentration of CPA in the anterior chamber over time.

FIG. 5: shows the results of an in vitro cornea permeability studyshowing the significant cornea permeability of CPA esters (Compound 2aand Compound 2g) respectively.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful toprovide definitions of certain terms to be used herein. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as is commonly understood by one of skill in the art to whichthis invention belongs.

DEFINITIONS

The term “cornea permeability” as used herein refers to the percentageof active compound delivered to the anterior chamber relative to thepercentage of a prodrug or active compound that is delivered topicallyto the cornea in an ocular eye drop (30-50 μl) for human cornea.

The term “C₁-C₁₀ optionally branched aliphatic” as used herein refers toa straight or branched chain; optionally unsaturated hydrocarbon havingfrom 1 to 10 carbon atoms. Representative C₁-C₁₀ aliphatic groupsinclude, but are not limited to ethylene, isopropylene, propyne, butyne,sec-butylene, pentylene, hexyldiene, heptylene, heptyne, octylene,octyne.

The term “aryl” as used herein refers to a phenyl group or a naphthylgroup. In one embodiment, the aryl group is substituted with one or moreof the following groups: —OH or OH—C₁-C₆alkyl groups. Unless indicated,the aryl is unsubstituted.

The term “C₃-C₈ cycloalkyl” as used herein is a 3-, 4-, 5-, 6-, 7- or8-membered saturated non-aromatic monocyclic cycloalkyl ring.Representative C₃-C₈ monocyclic cycloalkyl groups include, but are notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl and norbornyl. In one embodiment, the C₃-C₈monocyclic cycloalkyl group is substituted with one or more of thefollowing groups: —OH or OH—C₁-C₆alkyl groups. Unless indicated, theC₃-C₈ monocyclic cycloalkyl is unsubstituted.

The term “effective amount” as used herein refers to an amount of a CPAor a CPA prodrug that is effective for: (i) treating or preventingelevated IOP; or (ii) reducing IOP in a human.

The term “halo” as used herein refers to —F, —Cl, —Br or —I.

The term “C₃- to C₇-heterocyclic” refers to: (i) a 3- or 4-carbonmembered non-aromatic monocyclic cycloalkyl in which 1 of the ringcarbon atoms has been replaced with an N, O or S atom; or (ii) a 5-, 6-,or 7-carbon membered aromatic or non-aromatic monocyclic cycloalkyl inwhich 1-4 of the ring carbon atoms have been independently replaced witha N, O or S atom. The non-aromatic 3- to 7-carbon membered monocyclicheterocycles can be attached via a ring nitrogen, sulfur, or carbonatom. The aromatic 3- to 7-carbon membered monocyclic heterocycles areattached via a ring carbon atom. Representative examples of a C₃- toC₇-membered heterocyclic group include, but are not limited to furanyl,furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, isothiazolyl,isoxazolyl, morpholinyl, oxadiazolyl, oxazolidinyl, oxazolyl,oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,piperazinyl, piperidinyl, pyranyl, pyrazinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole,pyridothiazole, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl,quinuclidinyl, tetrahydrofuranyl, thiadiazinyl, thiadiazolyl, thienyl,thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiomorpholinyl,thiophenyl, triazinyl, triazolyl. In one embodiment, the C₃- toC₇-membered heterocyclics substituted with one or more of the followinggroups: OH or OH—C₁-C₆alkyl groups. Unless indicated, the 3- to7-membered monocyclic heterocycle is unsubstituted.

The phrase “pharmaceutically acceptable salt,” as used herein, is a saltof an acid and a basic nitrogen atom of a purine compound. Illustrativesalts include, but are not limited, to sulfate, citrate, acetate,oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acidphosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate,oleate, tannate, pantothenate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. The pharmaceuticallyacceptable salt can also be a camphorsulfonate salt. The term“pharmaceutically acceptable salt” also refers to a salt of a purinecompound having an acidic functional group, such as a carboxylic acidfunctional group, and a base. Suitable bases include, but are notlimited to, hydroxides of alkali metals such as sodium, potassium, andlithium; hydroxides of alkaline earth metal such as calcium andmagnesium; hydroxides of other metals, such as aluminum and zinc;ammonia, and organic amines, such as unsubstituted orhydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine;tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine;triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such asmono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-loweralkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine ortri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such asarginine, lysine, and the like. The term “pharmaceutically acceptablesalt” also includes a hydrate of a purine compound. Some chemicalstructures herein are depicted using bold and dashed lines to representchemical bonds. These bold and dashed lines depict absolutestereochemistry. A bold line indicates that a substituent is above theplane of the carbon atom to which it is attached and a dashed lineindicates that a substituent is below the plane of the carbon atom towhich it is attached.

This invention also encompasses pharmaceutical compositions containing,and methods of treating disorders through administering,pharmaceutically acceptable prodrugs of compounds of the invention. Forexample, compounds of the invention having free amino, amido, hydroxy orcarboxylic groups can be converted into prodrugs. Prodrugs includecompounds wherein an amino acid residue, or a polypeptide chain of twoor more (e.g., two, three or four) amino acid residues is covalentlyjoined through an amide or ester bond to a free amino, hydroxy orcarboxylic acid group of compounds of the invention. The amino acidresidues include but are not limited to the 20 naturally occurring aminoacids commonly designated by three letter symbols and also includes4-hydroxyproline, hydroxylysine, demosine, isodemosine,3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid,citrulline homocysteine, homoserine, ornithine and methionine sulfone.Additional types of prodrugs are also encompassed. For instance, freecarboxyl groups can be derivatized as amides or alkyl esters. Freehydroxy groups may be derivatized using groups including but not limitedto hemisuccinates, phosphate esters, dimethylaminoacetates, andphosphoryloxymethyloxycarbonyls, as outlined in Advanced Drug DeliveryReviews, 1996, 19, 115. Carbamate prodrugs of hydroxy and amino groupsare also included, as are carbonate prodrugs, sulfonate esters andsulfate esters of hydroxy groups. Derivatization of hydroxy groups as(acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group may bean alkyl ester, optionally substituted with groups including but notlimited to ether, amine and carboxylic acid functionalities, or wherethe acyl group is an amino acid ester as described above, are alsoencompassed. Prodrugs of this type are described in J. Med. Chem. 1996,39, 10. Free amines can also be derivatized as amides, sulfonamides orphosphonamides. All of these prodrug moieties may incorporate groupsincluding but not limited to ether, amine and carboxylic acidfunctionalities.

The following abbreviations are used herein and have the indicateddefinitions: CPA is N6-cyclopentyladenosine; NMR is nuclear magneticresonance; OHT is ocular hypertension or POAG is primary open-angleglaucoma.

The effects of CPA at lowering IOP have been previously reported inanimal models. However, the results reported are mixed.

In 1994, Crosson and Gray reported in the J. of Ocular Pharm. andTherapeutics. 10(1) 379-383 that the administration of CPA (165 μg)resulted in a reduction of rabbit IOP.

In 2001, Avila et al., reported in Brit. Journal of Pharmacology, (2001)134, 241-245, that the mouse is potentially a powerful vehicle forstudying pharmacology of aqueous humor dynamics, particularly in view ofthe increasing availability of knockout animals. In their studies Avilaet al. topically applied CPA using DMSO to the study eye of the mouse.They reported that the A₁ agonist CPA at 100 nm in three mice produced achange in IOP of −6.8 mm Hg (±1.8) while CPA at 1 mM in three miceproduced a change in IOP of −1.0 mm Hg (±2.3). Avila. et al. suggestedthat at increased concentrations CPA did not lower IOP because of anegative oculotensive effect of the A1 receptor offset by the opposingeffects of the A₃ and possibly A_(2A) receptors.

In 2003 Fleischhauer et al. reported results of a study in the J. ofMembrane Biol. (193, 121-36) where CPA was used to stimulate A₁adenosine receptors in isolated human trabecular meshwork (TM) cells.The trabecular meshwork is an area of ocular tissue located around thebase of the cornea, and is responsible for draining the aqueous humorfrom the eye via the anterior chamber (the chamber on the front of theeye covered by the cornea).

The authors concluded that the trabecular meshwork cells expressfunctional A₁, A_(2A) and A₃ receptors and that the enhancement ofaqueous humor outflow by A₁ adenosine receptor agonists, such as CPA maypossibly be mediated by reduction of TM cell volume.

In 2004 Hirao et al. reported in Experimental Eye Research 79, 729-735the results of studies that suggest that CPA at a concentration of 10nmol increased the optic nerve head tissue blood flow in rabbits afterCPA was injected intravitreally. The results also suggest that adenosineincreases the capillary blood flow in the optic nerve head of rabbits,and it acts through the A₁ and A_(2a) receptors from the ablumenal sidewhere pericytes are located.

In 2009, Dalpiaz et al. in Journal of Pharmaceutical Sciences, pages1-13 reported the preparation of a nanoparticle loaded with CPA. The CPAloaded nanoparticle was tested under in-vitro conditions and found topenetrate the cellular membrane of human retinal pigment endotheliumcells. As the authors describe in this paper, the clinical use of CPA ishampered by several aspects, including the fact that CPA is greatlyunstable in physiological fluids along with the potential forindiscriminate activity because of the fact that adenosine receptors areubiquitous in the body.

The inventors have also identified a number of CPA ester prodrugs thatdeliver CPA through the cornea. While some CPA esters have beenpreviously identified by Dalpiaz et al. in Pharmaceutical Research, Vol.18, No. 4, 2001 as being suitable as CPA prodrugs, there is nosuggestion that such prodrugs could be topically delivered onto thecornea so as to deliver CPA to the anterior chamber of a human subjectas a means of lowering the subject's IOP.

Traditional techniques of delivering CPA across the cornea of animalshave used dimethylsulfoxide (DMSO) as the carrier of CPA, however, it isthought that the DMSO has probably masked the impermeability of CPAbecause it is likely that DMSO disrupts the cornea and CPA is deliveredacross the cornea as a result of the disruption to the cornea. DMSO isnot a safe or suitable ocular solvent for human ocular drug delivery.

Surprisingly the inventors have found that the topical ocularadministration of compound A

in humans in a clinical trial resulted in the detection of CPA as anactive metabolite in conjunction with Compound A to lower IOP in humans.Co-pending applications U.S. Ser. No. 61/174,655 and U.S. Ser. No.61/219,990 teach the use of compound A in clinical trials to lower IOPin humans. The disclosures of U.S. Ser. No. 61/174,655 and U.S. Ser. No.61/219,990 are incorporated herein in their entirety.

The inventors have surprisingly found that CPA does not have sufficientcornea permeability to allow the topical delivery of safe levels of CPAto the cornea of a human subject. Furthermore, the inventors haveadditionally found that if an effective amount of CPA can be safelydelivered across the cornea of a human subject the subject's IOP can besignificantly reduced.

Embodiments of the present invention provide the use of CPA or CPAprodrugs for treating reducing and controlling normal or elevatedintraocular pressure (IOP) and/or treating glaucoma in human subjects.

Adenosine is a purine nucleoside that modulates many physiologicprocesses. Cellular signaling by adenosine occurs through four adenosinereceptor subtypes: A₁, A_(2A), A_(2B), and A₃ as reported by Ralevic andBurnstock (Pharmacol Rev. 50:413-492, 1988) and Fredholm B B et al.(Pharmacol Rev. 53:527-552, 2001). In the eye, adenosine A₁ receptoragonists lower IOP in mice, rabbits and monkeys (Tian B et al. Exp EyeRes. 64:979-989, 1997; Crosson C E. J Pharmacol Exp Ther. 273: 320-326,1995; and Avila M Y et al. Br J Pharmacol. 134:241-245, 2001). Whileother publications have noted that adenosine A1 receptor agonists in theeye target the conventional outflow pathway via the trabecular meshwork(Husain S et al. J Pharmacol Exp Ther. 320: 258-265, 2007), reduction ofIOP via other pathways has not been excluded.

It should be noted that the highly robust, adenosine A₁receptor-mediated drop in IOP reported in preclinical studies is oftenpreceded by an immediate, yet transient elevation in IOP followinginstillation of the A1 receptor ligand (Crosson C E and Grey T. InvOphthal Visual Sci. 37, [9] 1833-1839, 1996). Transient elevations inIOP of ˜3-9 mmHg have been observed in a ˜30 min “window” after dosing.This phenomenon may arise from cross-reactivity between adenosinereceptor sub-types within the eye. Pharmacological studies indicate thatthis transient elevation in IOP might be due, at least in part, to theactivation of adenosine A_(2B) receptors (Crosson, 1996). Therefore,development of a highly-selective A1 agonist that only reduce IOP wouldappear to be more tenable than the development of adenosineA2-receptor-based drugs for treating IOP, as A2A agonists may increase,decrease or exert mixed effects on IOP (Konno, 2004; Konno, J PharmacolSci., 2005; Konno, Eur J Pharmacol. 2005).

Compounds that act as selective adenosine A1 agonists are known and haveshown a variety of utilities. U.S. Pat. No. 7,423,144 to Jagtap et al.describes such selective adenosine A1 agonists compounds for theprevention or treatment of tachyarrhythmias (elevated heart rate), paindisorders, and ischemia-reperfusion injury.

It has now been found that CPA has been identified as an activemetabolite in clinical studies after the topical administration ofCompound A to the cornea of human subjects. The IOP of the humansubjects continues to decline after the buildup of CPA in the plasma ofthe human subjects and that no transient elevation in IOP is seensuggesting that the selectivity of CPA over the A₂ and A₃ adenosinereceptors is significant enough to avoid any transient increase in IOP.As shown in FIG. 2, the topical administration of Compound A to thecornea (see FIG. 1) of a human subject was found to result in thedetection of CPA in the plasma of a human subject, while the IOP of thesubject was still declining.

To further support the finding that CPA was arising in the plasma ofhuman subject after topical administration of Compound A to the cornea,additional in-vitro animal studies have been completed that show resultsas seen in FIG. 3 whereby topically applied Compound A to a corneamembrane resulted in the detection of significant levels of CPA on theother side of the cornea membrane. The same model was used to determinethe levels of CPA that could be transported across the cornea membraneand the results depicted in FIG. 3 show that the level of CPAtransported across the membrane is much less than that detected whenCompound A is topically applied to the cornea.

CPA or the compounds according to Formula II can be incorporated intovarious types of ophthalmic compositions or formulations for delivery.Formula I compounds may be delivered directly to the eye in acornea-permeable form (for example: topical ocular drops or ointmentscontaining nanoparticles of CPA; or via slow release devices such aspharmaceutical drug delivery sponges implanted in the cul-de-sac orimplanted adjacent to the sclera or within the eye; periocular,conjunctival, sub-tenons, intracameral, intravitreal, orintracanalicular injections). It is further contemplated that the agentsof the invention may be formulated in intraocular insert or implantdevices. It is envisaged that a nonaqueous nanoprecipitation techniquecould be used to form CPA-loaded nanoparticles having a particle size ofless than 0.25 μm (less than 250 nm). The corneal epithelial junctiongap has been measured by atomic force microscopy (AFM) as reported inThe Use of Atomic Force Microscopy for the Observation of CornealEpithelium Surface, Tsilimbaris et al., Investigative Ophthalmology &Visual Science, March 2000, Vol. 41, No. 3, pp. 680-686. A techniquesimilar to that described by Dalpiaz et al. in Journal of PharmaceuticalSciences, 2009, pages 1-13 would be suitable.

Formula II compounds may be delivered directly to the eye in acornea-permeable form (for example: topical ocular drops or ointments;or via slow release devices such as pharmaceutical drug delivery spongesimplanted in the cul-de-sac or implanted adjacent to the sclera orwithin the eye; periocular, conjunctival, sub-tenons, intracameral,intravitreal, or intracanalicular injections). It is furthercontemplated that the agents of the invention may be formulated inintraocular insert or implant devices. The compounds of Formula II arepreferably incorporated into topical ophthalmic formulations with a pHof about 4-8 for delivery to the eye. The compounds may be combined withophthalmologically acceptable preservatives, surfactants, viscosityenhancers, penetration enhancers, buffers, sodium chloride, and water toform an aqueous, sterile ophthalmic suspension or solution. Ophthalmicsolution formulations may be prepared by dissolving a compound in aphysiologically acceptable isotonic aqueous buffer. Further, theophthalmic solution may include an ophthalmologically acceptablesurfactant to assist in dissolving the compound. Furthermore, theophthalmic solution may contain an agent to increase viscosity orsolubility such as hydroxypropyl β-Cyclodextrin (HPβCD),hydroxymethylcellulose, hydroxyethylcellulose,hydroxypropylmethylcellulose, methylcellulose, polyvinylpyrrolidone, orthe like, to improve the retention of the formulation in theconjunctival sac. Gelling agents can also be used, including, but notlimited to, gellan and xanthan gum. In order to prepare sterileophthalmic ointment formulations, the active ingredient may be combinedwith a preservative in an appropriate vehicle such as mineral oil,liquid lanolin, or white petrolatum. Sterile ophthalmic gel formulationsmay be prepared by suspending the compound in a hydrophilic baseprepared from the combination of, for example, carbopol-974, or thelike, according to the published formulations for analogous ophthalmicpreparations; preservatives and tonicity agents can be incorporated.

Compounds in preferred embodiments are contained in a composition inamounts sufficient to lower IOP in patients experiencing elevated IOPand/or maintaining normal IOP levels in POAG or OHT patients. Suchamounts are referred to herein as “an amount effective to control orreduce IOP, “or more simply” an effective amount.” The compounds willnormally be contained in these formulations in an amount 0.05 mg/ml to7.0 mg/ml but preferably in an amount of 0.4 to 7.0 mg/ml. Thus, fortopical presentation 1 to 2 drops of these formulations would bedelivered to the surface of the eye from 1 to 4 times per day, accordingto the discretion of a skilled clinician. CPA or the compounds ofFormula II can also be used in combination with other glaucoma treatmentagents, such as, but not limited to, β-blockers, prostaglandin analogs,prostamides, carbonic anhydrase inhibitors, α₂ agonists, miotics, andneuroprotectants, ion channel modulators, A₁ agonists, A₃ antagonists,A₂A agonists and combinations thereof.

Any reference to a compound of the present invention is therefore to beunderstood as referring also to the corresponding pro-drugs of thecompound of the present invention, as appropriate and expedient.

In a first aspect, the present invention provides a method of reducingintraocular pressure comprising the step of: delivering an effectiveamount of cyclopentyladenosine according to Formula I to the anteriorchamber of an affected eye of a human,

with the proviso that the compound of Formula I is not delivered in theform of compound A

((2R,3S,4R,5R)-5-(6-(cyclopentylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methylnitrate

In one embodiment, the method comprises delivering an effective amountof the compound of Formula I, or a pharmaceutically acceptable salt of,to the anterior chamber of an affected eye of a human.

In another embodiment, the method comprises delivering an effectiveamount of a pharmaceutical composition comprising the compound ofFormula I to the anterior chamber of an affected eye of a human.

In another embodiment, the method comprises delivering an effectiveamount of a pharmaceutical composition comprising the compound ofFormula I, or a pharmaceutically acceptable salt of, to the anteriorchamber of an affected eye of a human.

In one embodiment the method comprises the step of applying about 0.05mg/ml to about 7.0 mg/ml of a compound according to Formula I from 1 to4 times daily, or in another embodiment the method comprises the step ofapplying about 20-700 μg of a compound according to Formula I from 1 to2 times daily or in another embodiment the method comprises the step ofapplying about 350 μg of a compound according to Formula I from 1 to 2times daily.

In one embodiment the IOP of the affected eye is reduced by at least10%. In another embodiment the IOP of the affected eye is reduced by atleast 10-20%.

In a further embodiment the IOP of the affected eye is reduced by 20% ormore.

In one embodiment the IOP of the affected eye is reduced by at least 10%for more than 3 hours, in another embodiment the IOP of the affected eyeis reduced by at least 10-20% for more than 3 hours, in a furtherembodiment the IOP of the affected eye is reduced by 20% or more formore than 3 hours and in another embodiment the IOP of the affected eyeis reduced by at least 10% for at least 6 hours.

In yet another aspect, the present invention provides a method ofreducing intraocular pressure comprising the step of: delivering aneffective amount of cyclopentyladenosine according to Formula I in acornea-permeable form to the anterior chamber of an affected eye of ahuman:

or a pharmaceutically acceptable salt thereof,

with the proviso that the compound of Formula I is not delivered in theform of compound A

((2R,3S,4R,5R)-5-(6-(cyclopentylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methylnitrate

In one embodiment the method comprises the step of applying about 0.05mg/ml to about 7.0 mg/ml of a compound according to Formula I from 1 to4 times daily, or in another embodiment the method comprises the step ofapplying about 20-700 μg of a compound according to Formula I from 1 to2 times daily or in another embodiment the method comprises the step ofapplying about 350 μg of a compound according to Formula I from 1 to 2times daily.

In one embodiment the IOP of the affected eye is reduced by at least10%. In another embodiment the IOP of the affected eye is reduced by atleast 10-20%.

In a further embodiment the IOP of the affected eye is reduced by 20% ormore.

In one embodiment the IOP of the affected eye is reduced by at least 10%for more than 3 hours, in another embodiment the IOP of the affected eyeis reduced by at least 10-20% for more than 3 hours, in a furtherembodiment the IOP of the affected eye is reduced by 20% or more formore than 3 hours and in another embodiment the IOP of the affected eyeis reduced by at least 10% for at least 6 hours.

In one embodiment the cornea-permeable form may be achieved by (i)delivering cornea-permeable nanoparticles of CPA.

In one embodiment the cornea-permeable nanoparticles of CPA are lessthan or about 200 nm.

In another aspect the present invention is directed to a compound ofFormula II,

or a pharmaceutically acceptable salt thereof,

wherein R₁ is selected from —(CO)C₁-C₆ alkyl, —(CO)CH(halo)₂,—(CO)phenyl, or a-(CO)C₁-C₁₀ optionally branched aliphatic, —(CO)C₃-C₈cycloalkyl which is optionally substituted with one or more of hydroxyor —(CH₂)_(n)—OH where n is 1-6, —(CO)aryl which is optionallysubstituted with one or more of hydroxy or —(CH₂)_(n)OH where n is 1-6;or a —(CO) C₃-C₇ heterocyclic which is optionally substituted with oneor more of hydroxy or —(CH₂)_(n)OH, where n is 1-6; R₂ is selected from—H or halo; and R₃ is selected from —H, hydroxy, —O(CO)CH(halo)₂,—O(CO)(CH₂)₂CH₃, —O(CO)CH(CH₃)₂, —O(CO)CH₂C(CH₃)₃.

In another embodiment R₁ is selected from —(CO)CH(CH₃)₂,—(CO)CH₂C(CH₃)₃, —(CO)C(CH₃)₃, —(CO)(CH₂)₂CH₃, —(CO)CH₂CH₃, —(CO)phenyl,or a-(CO)C₁-C₁₀ optionally branched aliphatic, —(CO)C₃-C₈ cycloalkylwhich is optionally substituted with one or more of hydroxy or—(CH₂)_(n)OH where n is 1-6, —(CO)aryl which is optionally substitutedwith one or more of hydroxy or —(CH₂)_(n)OH where n is 1-6; or a —(CO)C₃-C₇ heterocyclic which is optionally substituted with one or more ofhydroxy or —(CH₂)_(n)OH where n is 1-6; R₂ is selected from —H or halo;and R₃ is —H.

In one embodiment the compound of Formula II has the followingstructure:

or a pharmaceutically acceptable salt thereof.

In one embodiment the compound of Formula II has the followingstructure:

or a pharmaceutically acceptable salt thereof.

In another embodiment R₁ is selected from —(CO)CH(CH₃)₂ or—(CO)(CH₂)₆CH₃.

In another embodiment R₂ is —H.

In still another aspect the invention is directed to a method ofreducing IOP and associated diseases and conditions caused by elevatedIOP in a human subject by administering an effective amount of acompound of Formula II to an affected eye of the human subject:

or a pharmaceutically acceptable salt thereof,

wherein R₁ is selected from —(CO)C₁-C₆ alkyl, —(CO)CH(halo)₂,—(CO)phenyl, or a-(CO)C₁-C₁₀ optionally branched aliphatic, —(CO)C₃-C₈cycloalkyl which is optionally substituted with one or more of hydroxyor —(CH₂)_(n)OH where n is 1-6, —(CO)aryl which is optionallysubstituted with one or more of hydroxy or —(CH₂)_(n)OH where n is 1-6;or a —(CO) C₃-C₇ heterocyclic which is optionally substituted with oneor more of hydroxy or —(CH₂)_(n)OH where n is 1-6; R₂ is selected from—H or halo; and R₃ is selected from —H, hydroxy, —O(CO)CH(halo)₂,—O(CO)(CH₂)₂CH₃, —O(CO)CH(CH₃)₂, —O(CO)CH₂C(CH₃)₃.

In one embodiment of the method defined above, the compound of FormulaII has the following structure:

or a pharmaceutically acceptable salt thereof.

In a further embodiment of the method defined above, the compound ofFormula II has the following structure:

or a pharmaceutically acceptable salt thereof.

In one embodiment R₁ is selected from —(CO)CH(CH₃)₂, —(CO)CH₂C(CH₃)₃,—(CO)C(CH₃)₃, —(CO)(CH₂)₂CH₃, —(CO)CH₂CH₃, —(CO)phenyl, or a-(CO)C₁-C₁₀optionally branched aliphatic, —(CO)C₃-C₈ cycloalkyl which is optionallysubstituted with one or more of hydroxy or —(CH₂)_(n)OH where n is 1-6,—(CO)aryl which is optionally substituted with one or more of hydroxy or—(CH₂)_(n)OH where n is 1-6; or a —(CO) C₃-C₇ heterocyclic which isoptionally substituted with one or more of hydroxy or —(CH₂)_(n)OH wheren is 1-6; and R₂ is selected from —H or halo.

In one embodiment R₁ is selected from —(CO)CH(CH₃)₂, —(CO)(CH₂)₆CH₃,—(CO)CH₂C(CH₃)₃, —(CO)(CH₂)₃CH₃, (CO)C(CH₃)₃, (CO)(CH₂)₂CH₃, —(CO)CH₂CH₃or —(CO)phenyl to an affected eye of the subject.

In one embodiment R₂ is chloro.

In another embodiment, the compound of Formula II is Compound 2a.

In still another embodiment, the compound of Formula II is Compound 2g.

In one aspect, provided herein is the use of a compound according toFormula I for the manufacture of a medicament for reducing intraocularpressure.

In another aspect, provided herein is the use of a compound according toFormula II for the manufacture of a medicament for the treatment ofelevated IOP and diseases and conditions caused by elevated IOP.

In another aspect, provided herein is the use of compound 2a for themanufacture of a medicament for the treatment of elevated IOP anddiseases and conditions caused by elevated IOP.

In yet another aspect, provided herein is the use of compound 2g for themanufacture of a medicament for the treatment of elevated IOP anddiseases and conditions caused by elevated IOP.

Synthesis

The CPA 5′esters were prepared according to the following procedureshown in Scheme 1 below:

General Experimental Procedure

To a solution of 2′,3′-isopropylidene-N⁶-cyclopentyladenosine 1 (1.125g, 3 mmol) and DMAP (1.08 g, 9 mmol) in dichloromethane (15 mL) wasadded the corresponding acid chlorides drop wise and the reactionmixture was stirred at room temperature for 1 h. The reaction mixturewas then diluted with dichloromethane (10 mL) and washed with water(three times) and brine. The organic layer was separated, dried onsodium sulphate and concentrated on rotavaporator. The crude productobtained from the concentration was used as such in next step. A mixtureof water (8 mL) and TFA (2 mL) was slowly added to the crude product at0° C. and then the mixture was stirred at room temperature for 2 h. Itwas concentrated on rotavaporator and purified on pre-HPLC to get thedesired product 2.

N⁶-Cyclopentyl-5′-O-isobutyryl-adenosine (2a)

¹H NMR (CDCl₃): δ 1.04 (d, J=, 6.6 Hz, 3H), 1.08 (d, J=7.2 Hz, 3H),1.54-1.58 (m, 3H), 1.68-1.77 (m, 4H), 2.12 (m, 2H), 2.44-2.48 (m, 1H),4.22-4.28 (m, 1H), 4.36-4.39 (m, 4H), 4.47-4.52 (m, 2H), 5.91 (d, J=5.4Hz, 1H), 7.26 (s, 1H), 7.91 (s, 1H), 8.29 (s, 1H); (M+1): 406.3, Rt:4.9.

N⁶-Cyclopentyl-5′-O-(3-methylbutanoyl)-adenosine (2b)

¹H NMR (CDCl₃): δ 0.87 (d, J=, 4.5 Hz, 6H), 1.52-1.50 (m, 3H), 1.68-1.77(m, 4H), 1.95-2.0 (m, 1H), 2.10 (d, J=6.3 Hz, 4H), 4.22-4.30 (m, 1H),4.36-4.39 (m, 2H), 4.47-4.52 (m, 2H), 5.93 (d, J=4.8 Hz, 1H), 7.25 (s,1H), 7.92 (s, 1H), 8.29 (s, 1H).

N⁶-Cyclopentyl-5′-O-(2,2-dimethylpropanoyl)-adenosine (2c)

¹H NMR (CDCl₃): δ 1.09 (s, 9H), 1.52-1.50 (m, 3H), 1.68-1.77 (m, 4H),2.13 (d, J=5.7 Hz, 3H), 3.64 (s, 1H), 4.22-4.29 (m, 1H), 4.34-4.39 (m,2H), 4.47-4.52 (m, 2H), 5.93 (d, J=5.7 Hz, 1H), 7.25 (s, 1H), 7.90 (s,1H), 8.28 (s, 1H).

N⁶-Cyclopentyl-5′-O-propanoyl-adenosine (2d)

¹H NMR (CDCl₃): δ 1.05 (t, J=7.5 Hz, 3H), 1.52-1.50 (m, 3H), 1.68-1.77(m, 4H), 2.13 (d, J=5.7 Hz, 3H), 2.24-2.27 (m, 2H), 3.62 (s, 1H),4.22-4.29 (m, 1H), 4.34-4.39 (m, 2H), 4.47-4.52 (m, 2H), 4.58 (s, 1H),5.94 (s, 1H), 7.25 (s, 1H), 7.91 (s, 1H), 8.28 (s, 1H).

N⁶-Cyclopentyl-5′-O-butanoyl-adenosine (2e)

¹H NMR (CDCl₃): δ 0.86 (t, J=7.2 Hz, 3H), 1.52-1.59 (m, 4H), 1.68-1.77(m, 4H), 2.11-2.22 (m, 6H), 3.65 (s, 1H), 4.22-4.29 (m, 1H), 4.34-4.39(m, 2H), 4.47-4.52 (m, 2H), 4.58 (s, 1H), 5.93-5.94 (m, 2H), 7.26 (s,1H), 7.91 (s, 1H), 8.28 (s, 1H).

N⁶-Cyclopentyl-5′-O-pentanoyl-adenosine (2f)

¹H NMR (CDCl₃): δ 0.81 (t, J=7.2 Hz, 3H), 1.19-1.27 (m, 2H), 1.42-1.58(m, 6H), 1.68-1.77 (m, 3H), 1.97 (m, 1H), 2.12-2.22 (m, 5H), 3.51 (s,1H), 4.22-4.29 (m, 1H), 4.34-4.39 (m, 2H), 4.47-4.52 (m, 2H), 4.59 (s,1H), 5.92 (d, J=5.4 Hz, 2H), 7.26 (s, 1H), 7.90 (s, 1H), 8.29 (s, 1H).

N⁶-Cyclopentyl-5′-O-octanoyl-adenosine (2g)

¹H NMR (CDCl₃): δ 0.83-0/87 (m, 3H), 1.19-1.30 (m, 9H), 1.46-1.79 (m,6H), 2.10-2.22 (m, 3H), 2.34 (dd, J=7.2 and 7.5 Hz, 1H), 4.22-4.29 (m,1H), 4.34-4.39 (m, 2H), 4.46-4.52 (m, 2H), 4.59 (s, 1H), 5.92 (d, J=5.4Hz, 2H), 7.26 (s, 1H), 7.93 (s, 1H), 8.30 (s, 1H), (M+1): 462.3.

N⁶-Cyclopentyl-5′-O-benzoyl-adenosine (2h)

¹H NMR (CDCl₃): δ 1.52-1.58 (m, 2H), 1.68-1.76 (m, 3H), 2.10-2.20 (m,3H), 3.7 (s, 1H), 4.49-4.54 (m, 2H), 4.60-4.65 (m, 4H), 5.94 (d, J=4.8Hz, 2H), 7.25-7.33 (m, 3H), 7.46-7.49 (m, 1H), 7.81 (d, J=6.9 Hz, 2H),7.89 (s, 1H), 8.24 (s, 1H) The N⁶ hydroxy or N⁶ esters substituted CPAesters were prepared according to the procedure shown in Scheme 2 below:

((2R,3S,4R,5R)-3,4-Dihydroxy-5-(6-((1R,2R)-2-hydroxycyclopentylamino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl3,3-dimethylbutanoate (3a)

¹HNMR (DMSO-d6): 0.80-0.84 (m, 1H), 0.89 (s, 9H), 1.42-1.65 (m, 3H),1.80-2.02 (m, 3H), 3.14 (s, 2H), 4.01-4.27 (m, 4H), 4.66 (s, 1H), 4.86(s, 1H), 5.38 (s, 1H), 5.58 (s, 1H), 5.88 (d, J=5.1 Hz, 1H), 7.24 (bs,1H), 7.67 (d, J=6.6 Hz, 1H), 8.18 (s, 1H), 8.28 (s, 1H).

((2R,3S,4R,5R)-5-(6-((1R,2R)-2-(2,2-Dichloroacetoxy)cyclopentylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl2,2-dichloroacetate (3b)

¹HNMR (DMSO-d6): 0.80-0.84 (m, 1H), 1.07-1.12 (m, 2H), 1.64-1.20 (m,2H), 2.10-2.13 (m, 2H), 4.13 (s, 1H), 4.24 (s, 1H), 4.40-4.62 (m, 3H),5.26 (s, 1H), 5.43 (s, 1H), 5.60 (d, J=4.8 Hz, 1H), 5.92 (d, J=4.8 Hz,1H), 6.82 (s, 1H), 6.90 (s, 1H), 8.05 (s, 1H), 8.19 (s, 1H), 8.31 (s,1H).

((2R,3S,4R,5R)-3,4-Dihydroxy-5-(6-((1R,3R)-3-(isobutyryloxy)cyclopentylamino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methylisobutyrate (3c)

MS (ES⁺): m/z 392.2 (M+1)

((2R,3S,4R,5R)-5-(6-((1R,3R)-3-(Butyryloxy)cyclopentylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methylbutyrate (3d)

MS (ES⁺): m/z 392.2 (M+1)

((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-((1S,2S)-2-(Isobutyryloxy)cyclopentylamino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methylisobutyrate (3e)

MS (ES⁺): m/z 392.2 (M+1)

((2R,3S,4R,5R)-5-(6-((1S,2S)-2-(Butyryloxy)cyclopentylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methylbutyrate (3f)

MS (ES⁺): m/z 392.2 (M+1)

The 2′3′ esters defined above could be made according to the followinggeneral procedure.

Example I In-Vitro Cornea Permeability Studies

With reference to FIG. 3, which shows an in-vitro system for measuringthe cornea permeability of a cornea the compounds 2a and 2g wereselected for study using dutch belted cornea membranes.

The compounds (2a) and (2g) were prepared in powder form by InotekPharmaceuticals Corp. (Lexington, Mass.). Low-permeability controlcompound atenolol and all other chemicals were purchased from Sigma (St.Louis, Mo.). The buffer used in the permeability assessment was aglutathione-bicarbonated Ringer's (GBR) solution (110 mM NaCl, 5 mM KCl,1 mM NaH2PO4, 30 mM NaHCO₃, 1 mM CaCl₂, 0.75 mM Mg Cl₂, 5 mM D-glucose,and 0.3 mM reduced glutathione), pH 7.4, which was freshly prepared onthe day of the experiment and oxygenated with O₂/CO₂ (95:5) to pH 7.4.

The compounds (2a) and (2g) were reconstituted in saline and diluted(10-fold) into the assay at a final concentration between 50 μM and 2.6mM. Male Dutch-belted pigmented rabbits (1.5-2.5 kg body weight, 3-3.5months old) were purchased from Covance Research Products Inc. (Denver,Pa.). The animal handling performed in this study conformed to theGuiding Principles in the Care and Use of Animals (DHEW Publication, NIH80-23). The rabbits were euthanized by CO₂ asphyxiation, and the headswere transported on ice to a testing facility, where dissection of theeyes was performed.

The corneal tissues were excised and mounted on a Harvard verticaldiffusion apparatus as shown in FIG. 3 with a diffusion area of 0.64cm2. Preheated (37° C.), pH 7.4, GBR buffer was added to the mucosal(1.5 mL) and the serosal (1.5 mL) chambers. The diffusion apparatus wasmaintained at 37° C. throughout the entire transport experiment.Oxygenation and agitation were achieved by bubbling O₂/CO₂ (95:5)through each chamber at a rate of 5-6 bubbles per second. After the30-minute equilibration, blank GBR buffer in the mucosal (donor) chamberwas withdrawn and replaced with GBR assay buffer containing the compound(2a) or the compound (2g). The transport experiments lasted 2 hours andwere performed in duplicate. Every 60 minutes, 0.2-mL samples werecollected from the serosal (receiver) chamber and replenished with0.2-mL blank GBR buffer, except at the last time point; at the end ofthe experiment, samples were also collected from the mucosal (donor)chambers for mass balance determination.

After the transport experiment, tissue integrity (system suitability)was assessed by measuring the permeation of a low permeability controlcompound, atenolol, across the tissue. Donor chamber contents werereplaced with GBR buffer containing 100 μM atenolol, and receiverchambers were replaced with fresh blank GBR buffer. After 30 minutes ofincubation, samples were collected from both chambers for analysis. Thepost-experimental system suitability assessment was consideredacceptable if duplicate measurements yielded a mean apparentpermeability (P_(app)) value for atenolol <1·10⁻⁶ cm/s.

Compound A and compounds (2a) and (2g) and atenolol concentrations inthe donor and receiver chambers were analyzed by LC-MS/MS methods.Apparent permeability (Papp) values were calculated using the followingequation:

P _(app)=(dC _(r) /dt)·V _(r)/(A·C ₀)

Where, dCr/dt was the slope of the linear portion of the cumulativeconcentration in the receiver compartment over time in μM/sec, V_(r) wasthe volume of the receiver chamber in cm³, A was the diffusion area incm², and C₀ was the measured dosing concentration in μM.

Recovery was calculated using the following equation:

Recovery=100·(V _(r) ·C _(r) ^(final) +V _(d) ·C _(d) ^(final))/(V _(d)·C ₀)

Where, V_(r) was the volume of the receiver compartment in cm³, V_(d)was the volume of the donor compartment in cm³, C₀ was the dosingconcentration in μM, C_(r) ^(final) was the cumulative receiverconcentration in μM, and C_(d) ^(final) was the donor concentration inμM at the end of the incubation.

It can be seen from the graphs shown in FIG. 5 that significant levelsof CPA were measureable when Compounds (2a) and (2g) were placed in thedonor chamber, providing support for the release of CPA from Compounds(2a) and (2g) after passage through a cornea membrane.

Example II Analysis of Human Plasma after Administration of Compound A

After the administration of Compound A topically to a cornea of a human,at selected time points (e.g., Day 1: pre-dose, 5, 15, 25, 35, and 45min and 1, 2, 4, 8 and 24 hours) samples of whole blood (10 mL) werecollected for pharmacokinetic assessments using a vacutainer tubecontaining sodium heparin as an anticoagulant, via catheter, salinelock, or by venipuncture. The blood components were separated bycentrifugation at 4° C. following standard clinical laboratoryprocedures for preparation of plasma from whole blood (e.g., 3000 rpmfor approximately 10 min). For each sample, approximately 1 mL of plasmawas stored at −20° C. or colder until analysis for Compound A and CPAconcentration. Human plasma samples were analyzed for Compound Aconcentrations using a validated liquid chromatography/tandem massspectrometry (LC/MS/MS) method with a lower limit of quantitation (LLOQ)of 10.0 pg/mL and a linear range from 10.0 to 2000 pg/mL. Plasmaconcentrations of (N(6)-cyclopentyladenosine, CPA) were also measured insome samples using a validated LC/MS/MS method with a lower limit ofquantitation of 10.0 pg/mL and a linear range from 10.0 to 2000 pg/mL.

As a result of this analysis, CPA has been identified as an activemetabolite in clinical studies after the topical administration ofCompound A to the cornea of human subjects. The IOP of the humansubjects continues to decline after the buildup of CPA in the plasma ofthe human subjects and that no transient elevation in IOP is seensuggesting that the selectivity of CPA over the A₂ and A₃ adenosinereceptors is significant enough to avoid any transient increase in IOP.As shown in FIG. 2, the topical administration of Compound A to thecornea (see FIG. 1) of a human subject was found to result in thedetection of CPA in the plasma of a human subject, while the IOP of thesubject was still declining.

The present invention and its embodiments have been described in detail.However, the scope of the present invention is not intended to belimited to the particular embodiments of any process, manufacture,composition of matter, compounds, means, methods, and/or steps describedin the specification. Various modifications, substitutions, andvariations can be made to the disclosed material without departing fromthe spirit and/or essential characteristics of the present invention.Accordingly, one of ordinary skill in the art will readily appreciatefrom the disclosure that later modifications, substitutions, and/orvariations performing substantially the same function or achievingsubstantially the same result as embodiments described herein may beutilized according to such related embodiments of the present invention.Thus, the following claims are intended to encompass within their scopemodifications, substitutions, and variations to processes, manufactures,compositions of matter, compounds, means, methods, and/or stepsdisclosed herein.

We claim:
 1. A method of reducing intraocular pressure comprising thestep of: delivering an effective amount of cyclopentyladenosine (CPA)according to Formula I, or a pharmaceutically acceptable salt thereof,to the anterior chamber of an affected eye of a human

with the proviso that the compound of Formula I is not delivered in theform of compound A


2. The method as claimed in claim 1 comprising the step of applyingabout 0.05 mg/ml to about 7.0 mg/ml of a compound according to Formula Ifrom 1 to 4 times daily.
 3. The method as claimed in claim 1 comprisingthe step of applying about 20-700 μg of a compound according to FormulaI from 1 to 2 times daily.
 4. The method as claimed in claim 1comprising the step of applying about 350 μg of a compound according toFormula I from 1 to 2 times daily.
 5. The method as claimed in claim 1wherein the IOP of the affected eye is reduced by at least 10%.
 6. Themethod as claimed in claim 1 wherein the IOP of the affected eye isreduced by at least 10-20%.
 7. The method as claimed in claim 1, whereinthe IOP of the affected eye is reduced by 20% or more.
 8. The method asclaimed in claim 1 wherein the IOP of the affected eye is reduced by atleast 10% for more than 3 hours.
 9. The method as claimed in claim 1wherein the IOP of the affected eye is reduced by at least 10-20% formore than 3 hours.
 10. The method as claimed in claim 1 wherein the IOPof the affected eye is reduced by 20% or more for more than 3 hours. 11.The method as claimed in claim 1, wherein the IOP of the affected eye isreduced by at least 10% for at least 6 hours.
 12. The method as claimedin claim 1, further comprising prior, simultaneous or sequential,application of a second IOP reducing agent.
 13. The method as claimed inclaim 12 wherein the second IOP reducing agent is selected from thegroup comprising: β-blockers, prostaglandin analogs, prostamides,carbonic anhydrase inhibitors, rho-kinase inhibitors, α₂ agonists,miotics, ion channel modulators, neuroprotectants, A₃ adenosine receptorantagonists, A_(2A) adenosine receptor agonists, and combinationsthereof.
 14. A method of reducing intraocular pressure comprising thestep of: delivering an effective amount of cyclopentyladenosine (CPA)according to Formula I, or a pharmaceutically acceptable salt thereof,in a cornea-permeable form to the anterior chamber of an affected eye ofa human

with the proviso that the compound of Formula I is not delivered in theform of compound A


15. The method as claimed in claim 14 comprising the step of applyingabout 0.05 mg/ml to about 7.0 mg/ml of a compound according to Formula Ifrom 1 to 4 times daily.
 16. The method as claimed in claim 14comprising the step of applying about 20-700 μg of a compound accordingto Formula I from 1 to 2 times daily.
 17. The method as claimed in claim14 comprising the step of applying about 350 μg of a compound accordingto Formula I from 1 to 2 times daily.
 18. The method as claimed in claim14 wherein the IOP of the affected eye is reduced by at least 10%. 19.The method as claimed in claim 14 wherein the IOP of the affected eye isreduced by at least 10-20%.
 20. The method as claimed in claim 14wherein the IOP of the affected eye is reduced by 20% or more.
 21. Themethod as claimed in claim 14 wherein the IOP of the affected eye isreduced by at least 10% for more than 3 hours.
 22. The method as claimedin claim 14 wherein the IOP of the affected eye is reduced by at least10-20% for more than 3 hours.
 23. The method as claimed in claim 14wherein the IOP of the affected eye is reduced by 20% or more for morethan 3 hours.
 24. The method as claimed in claim 14 wherein the IOP ofthe affected eye is reduced by at least 10% for at least 6 hours. 25.The method as claimed in claim 14 wherein the cornea-permeable form isachieved by delivering cornea-permeable nanoparticles of CPA.
 26. Themethod as claimed in claim 25 wherein the cornea-permeable nanoparticlesof CPA are at less than or about 200 nm.
 27. The method as claimed inclaim 14 wherein the method further comprises the prior, simultaneous orsequential, application of a second IOP reducing agent.
 28. The methodas claimed in claim 27 wherein second IOP reducing agent is selectedfrom the group comprising: β-blockers, prostaglandin analog,prostamides, carbonic anhydrase inhibitors, rho-kinase inhibitors, α₂agonists, miotics, ion channel modulators, neuroprotectants, A₁adenosine receptor agonists, A₃ adenosine receptor antagonists, A_(2A)adenosine receptor agonists, and combinations thereof.